Compositions and methods for controlling insects

ABSTRACT

The present invention comprises compositions, methods and cell lines related to controlling insects. An embodiment of a composition comprises a plant essential oil and targets at least one receptor of insects chosen from tyramine receptor, Or83b olfactory receptor, and Or43a olfactory receptor, resulting in a change in the intracellular levels of cAMP, Ca2+, or both in the insects.

CROSS REFERENCES TO RELATED APPLICATION

This application is a divisional of commonly assigned and co-pending U.S. patent application Ser. No. 10/832,022 filed Apr. 26, 2004, which claims priority from U.S. Provisional Application Ser. No. 60/465,320 filed Apr. 24, 2003 and U.S. Provisional Application Ser. No. 60/532,503 filed Dec. 24, 2003, which are both incorporated herein in their entirety by this reference. The entire disclosures contained in U.S. application Ser. No. 10/832,022 and U.S. Provisional Application Ser. Nos. 60/465,320 and 60/532,503 are incorporated herein by this reference.

FIELD OF THE INVENTION

The present invention relates to compositions, methods, cell lines and reports related to controlling insects.

BACKGROUND OF THE INVENTION

Animals have chemosensory and mechanosensory systems that recognize a large array of environmental stimuli, generating behavioral responses. Behavioral studies have been conducted to understand the genetics of these systems. The olfactory system plays a role in the survival and maintenance of species, including insects.

Drosophila is one of the models for studying the sensory system, as it is amenable to mutant analysis using molecular techniques, behavioral analysis, and electrophysiological analysis, and because its olfactory system is comparable to the mammalian counterpart.

Various chemicals and mixtures have been studied for pesticidal activity for many years with a goal of obtaining a product which is selective for invertebrates such as insects and has little or no toxicity to vertebrates such as mammals, fish, fowl and other species and does not otherwise persist in and damage the environment.

Most of the previously known and commercialized products having sufficient pesticidal activity to be useful also have toxic or deleterious effects on mammals, fish, fowl or other species which are not the target of the product. For example, organophosphorus compounds and carbamates inhibit the activity of acetylcholinesterase in insects as well as in all classes of animals. Chlordimeform and related formamidines are known to act on octopamine receptors of insects but have been removed from the market because of cardiotoxic potential in vertebrates and carcinogenicity in animals and a varied effect on different insects. Other compounds, which may be less toxic to mammals and other non-target species, are sometimes difficult to identify.

SUMMARY OF THE INVENTION

The present invention comprises compositions for controlling insects and methods for using these compositions. The present invention comprises compositions for controlling insects, which comprise one or more plant essential oils and methods for using these compositions. The plant essential oils, when combined, may have a synergistic effect. The compositions may include a fixed oil, which is a non-volitile non-scented plant oil. Additionally, it is contemplated that these compositions may be made up of generally regarded as safe (GRAS) compounds.

The present invention comprises compositions comprising one or more plant essential oils and an insect control agent, and methods for using these compositions. Examples of insect control agent include, DEET and D-allethrin. The plant essential oil and the insect control agent, when combined, may have a synergistic effect. For example, the insect control activity of 29% DEET may be achieved with 5% DEET when included in a combination of the present invention.

The present invention comprises a method for screening compositions for insect control activity. The present invention comprises cell lines stably transfected with tyramine receptor (TyrR), Or83b Olfactory Receptor (Or83b), or Or43a Olfactory Receptor, which may be used to screen compositions for insect control activity.

The present invention comprises a method for generating a report identifying one or more compositions having insect control activity. The term “report” refers to statements or descriptions contained in a printed document, a database, a computer system, or other medium.

For purposes of simplicity, the term “insect” shall be used through out this application; however, it should be understood that the term “insect” refers, not only to insects, but also to arachnids, larvae, and like invertebrates. Also for purposes of this application, the term “insect control” shall refer to having a repellant effect, a pesticidal effect, or both. “Repellant effect” is an effect, wherein more insects are repelled away from a host or area that has been treated with the composition than a control host or area that has not been treated with the composition. In some embodiments, repellant effect is an effect wherein at least about 75% of insects are repelled away from a host or area that has been treated with the composition. In some embodiments, repellant effect is an effect wherein at least about 90% of insects are repelled away from a host or area that has been treated with the composition. “Pesticidal effect” is an effect, wherein treatment with a composition causes at least about 1% of the insects to die. In this regard, an LC1 to LC100 (lethal concentration) or an LD1 to LD100 (lethal dose) of a composition will cause a pesticidal effect. In some embodiments, the pesticidal effect is an effect, wherein treatment with a composition causes at least about 5% of the exposed insects to die. In some embodiments, the pesticidal effect is an effect, wherein treatment with a composition causes at least about 10% of the exposed insects to die. In some embodiments, the pesticidal effect is an effect, wherein treatment with a composition causes at least about 25% of the insects to die. In some embodiments the pesticidal effect is an effect, wherein treatment with a composition causes at least about 50% of the exposed insects to die. In some embodiments the pesticidal effect is an effect, wherein treatment with a composition causes at least about 75% of the exposed insects to die. In some embodiments the pesticidal effect is an effect, wherein treatment with a composition causes at least about 90% of the exposed insects to die. In some embodiments of the invention, treatment with such concentrations or doses will result in a knockdown of the insects occurring within a few seconds to a few minutes.

The compositions of the present invention may be used to control insects by either treating a host directly, or treating an area in which the host will be located, for example, an indoor living space, outdoor patio or garden. For purposes of this application, host is defined as a plant, human or other animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the receptor-specific binding in Schneider cells transfected with the tyramine receptor;

FIG. 2 shows the saturation binding curve of ³H-tyramine in membranes prepared from Schneider cells expressing the tyramine receptor after incubation with ³H-tyramine at various concentrations in the presence or absence of unlabeled tyramine;

FIG. 3 shows the inhibition binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the tyramine receptor after incubation with ³H-tyramine in the presence and absence of different concentrations of the unlabeled tyramine;

FIG. 4 shows the inhibition binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the tyramine receptor in the presence and absence of different concentrations of the unlabeled ligands: tyramine (TA), octopamine (OA), dopamine (DA), and serotonin (SE);

FIG. 5 shows the Inhibition binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the tyramine receptor after incubation with ³H-tyramine in the presence and absence of different concentrations of Lilac Flower Oil (LFO) and Black Seed Oil (BSO);

FIG. 6 shows the inhibition binding of ³H-tyramine (3H-TA) to membranes prepared from Schneider cells expressing the tyramine receptor after incubation with ³H-tyramine in the presence and absence of either LFO or BSO or in combination with different concentrations of unlabeled tyramine (TA);

FIG. 7 shows tyramine dependent changes in cAMP levels in Schneider cells expressing the tyramine receptor in the presence and absence of forskolin and tyramine;

FIG. 8 shows tyramine dependent changes in cAMP levels in Schneider cells expressing the tyramine receptor treated with Lilac Flower Oil and Black Seed Oil in the presence and absence of forskolin and tyramine;

FIG. 9 shows tyramine dependent changes in cAMP levels in Schneider cells expressing the tyramine receptor after treatment with forskolin in the presence and absence of tyramine, Lilac Flower Oil and Black Seed Oil;

FIG. 10 shows the saturation binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the Or83b receptor;

FIG. 11 shows the saturation binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the Or 43a receptor;

FIG. 12 shows the forskolin-dependent changes in cAMP levels in Schneider cells expressing the Or83b receptor;

FIG. 13 shows the ionomycin-dependent changes in intracellular Ca²⁺ levels in Schneider cells expressing the Or83b receptor;

FIG. 14 shows the ionomycin-dependent changes in intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 15 shows the tyramine-dependent changes in intracellular Ca²⁺ levels in control Schneider cells, Schneider cells expressing the Or83b receptor, and Schneider cells expressing the Or 43a receptor;

FIG. 16 shows the interaction of various plant essential oils, including, LFO, piperonal, diethyl phthalate, and α-terpineol, with the Or83b and Or43a receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 17 shows the interaction of various plant essential oils, including, BSO, quinine, sabinene, α-thujone, α-pinene, d-limonene, and p-cymene with the Or43a receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 18 shows the interaction of various plant essential oils, including, BSO, quinine, sabinene, α-thujone, α-pinene, d-limonene, and p-cymene with the Or83b receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 19 shows the interaction of various plant essential oils, including, geraniol, linalyl anthranilate, phenyl acetaldehyde, linalool, α-terpineol, t-anethole, terpinene 900, lindenol, and eugenol, with the Or83b and Or43a receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 20 shows the interaction of various plant essential oils, including, thyme oil, carvacrol, and thymol, with the Or83b and Or43a receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 21 shows the interaction of various plant essential oils, including, piperonal, piperonyl alcohol, piperonyl acetate, and piperonyl amine, with the Or83b and Or43a receptors in Schneider cells expressing the olfactory receptors after incubation with ³H-tyramine;

FIG. 22 shows the effect of ionomycin, tyramin, and linalyl anthranilate on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 23 shows the effect of linalool, perillyl alcohol, t-anethole, geraniol, phenyl acetaldehyde, and eugenol on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 24 shows the effect of piperonyl, piperonyl alcohol, piperonyl acetate, and piperonyl amine on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 25 shows the effect of α-termineol, lindenol, and terpinene 900 on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 26 shows the effect of thyme oil, thymol, and carvacrol on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor;

FIG. 27 shows the effect of LFO on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor or the Or83b receptor;

FIG. 28 shows the effect of BSO, α-pinene, p-cymene, d-limonene, sabinene, quinine, l-carvone, d-carvone, and α-thujone on intracellular Ca²⁺ levels in Schneider cells expressing the Or43a receptor or the Or83b receptor;

FIG. 29 shows tyramine dependent changes in cAMP levels in Schneider cells expressing Or83b receptor in the presence and absence of tyramine, LFO and BSO;

FIG. 30 shows the tyramine dependent changes in cAMP levels in Schneider cells expressing Or83b receptor treated with LFO and BSO in the presence and absence of tyramine and forskolin;

FIGS. 31A and 31B show the nucleic acid sequence and the peptide sequence of tyramine receptor;

FIGS. 32A and 32B show the nucleic acid sequence and the peptide sequence of Or43a olfactory receptor; and

FIGS. 33A and 33B show the nucleic acid sequence and the peptide sequence of Or 83b olfactory receptor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions, methods, cell lines and reports related to controlling insects. The insect control may be related to one or more of the receptors, comprising tyramine receptor (TyrR), Or83b Olfactory receptor (Or83b), and Or43a olfactory receptor (Or43a).

The present invention comprises a method for screening compositions for insect control activity. The present invention comprises Drosophila Schneider cell lines stably transfected with TyrR, Or43a, or Or83b, which may be used to screen compositions for insect control activity. The nucleic acid sequence and the peptide sequence of TyrR are set forth in FIGS. 31A and 31B. The nucleic acid sequence and the peptide sequence of Or43a are set forth in FIGS. 32A and 32B. The nucleic acid sequence and the peptide sequence of Or83b are set forth in FIGS. 33A and 33B.

The potential for insect control activity may be identified by measuring the affinity of the test compositions for the receptor in the cell lines expressing TyrR, Or83b, and/or Or43a. The potential for insect control activity may also be identified by measuring the change in intracellular cAMP and/or Ca²⁺ in the cell lines expressing TyrR, Or83b, and/or Or43a following treatment with the test compositions. The gene sequences of the TyrR receptor, the Or 83b receptor and the Or 43a receptor have substantial similarity between various insect species. As such, the Drosophila Schneider cell lines expressing these receptors may be used to screen for compositions having insect control activity in various insect species.

The present invention comprises compositions for controlling insects and methods for using these compositions. The present invention comprises compositions for controlling insects, which comprise one or more plant essential oils and methods for using these compositions. The plant essential oils, when combined, may have a synergistic effect. The compositions of the present invention may include any of the following oils, or mixtures thereof:

t-anthole lime oil piperonyl Black seed oil (BSO) d-limonene piperonyl acetate camphene linalyl anthranilate piperonyl alcohol carvacrol linalool piperonyl amine d-carvone lindenol quinone 1-carvone methyl citrate sabinene 1,8-cineole methyl α-terpinene di-hydrojasmonate p-cymene myrcene terpinene 900 diethyl phthalate perillyl alcohol α-terpineol eugenol phenyl acetaldehyde gamma-terpineol geraniol α-pinene 2-tert-butyl-p-quinone isopropyl citrate β-pinene α-thujone lemon grass oil piperonal thyme oil lilac flower oil (LFO) thymol

The compositions of the present invention may also include any of the following oils, or mixtures thereof:

Allyl sulfide β-elemene Menthyl salicylate Allyl trisulfide gamma-elemene Myrtenal Allyl-disulfide Elmol Neraldimethyl acetate Anethole Estragole Nerolidol Artemisia alcohol 2-ethyl-2-hexen-1-ol Nonanone acetate Benzyl acetate Eugenol acetate 1-octanol Benzyl alcohol α-farnesene E ocimenone Bergamotene (Z,E)-α-farnesene Z ocimenone β-bisabolene E-β-farnesene 3-octanone Bisabolene oxide Fenchone Ocimene α-bisabolol Furanodiene Furanoeudesma- Octyl acetate Bisabolol oxide 1,3-diene Peppermint oil Bisobolol oxide β Furanoeudesma-1,4-diene α-phellandrene Bornyl acetate Furano germacra 1,10(15)- β-phellandrene β-bourbonene diene-6-one piperonal α-cadinol Furanosesquiterpene Prenal Camphene Geraniol Pulegone α-campholene Geraniol acetate Sabinene α-campholene Germacrene D Sabinyl acetate aldehyde camphor Germacrene B α-santalene Caryophyllene oxide α-gurjunene Santalol Chamazulene α-humulene Sativen Cinnamaldehyde α-ionone δ-selinene Cis-verbenol β-ionone β-sesquphelandrene Citral A Isoborneol Spathulenol Citral B Isofuranogermacrene Tagetone Citronellal Iso-menthone α-terpinene Citronellol Iso-pulegone 4-terpineol Citronellyl acetate Jasmone α-terpinolene Citronellyl formate Lilac flower oil α-terpinyl acetate α-copaene Limonene α-thujene cornmint oil Linalool Thymyl methyl ether β-costol Linalyl acetate Trans-caryophyllene Cryptone Lindestrene Trans-pinocarveol Curzerenone Methyl-allyl-trisulfide Trans-verbenol d-Carvone Menthol Verbenone l-Carvone 2-methoxy furanodiene Yomogi alcohol Davanone menthone Zingiberene Diallyl tetrasulfide Menthyl acetate Dihydrotagentone dihydro- Methyl cinnamate pyrocurzerenone

In those compositions including more than one oil, each oil may make up between about 1% to about 99%, by weight, of the composition mixture. For example, one composition of the present invention comprise about 1% thymol and about 99% geraniol. Optionally, the compositions may additionally comprise a fixed oil, which is a non-volitile non-scented plant oil. For example, the composition could include one or more of the following fixed oils:

castor oil mineral oil safflower oil corn oil olive oil sesame oil cumin oil peanut oil soy bean oil For example, one composition of the present invention includes about 1% thymol, about 50% geraniol and about 49% mineral oil. Additionally, it is contemplated that these compositions may be made up of generally regarded as safe (GRAS) compounds, for example: thyme oil, geraniol, lemon grass oil, lilac flower oil, black seed oil, lime oil, eugenol, castor oil, mineral oil, and safflower oil.

The present invention comprises compositions comprising one or more plant essential oils and an insect control agent, for example, DEET, and D-allethrin, and methods for using these compositions. The plant essential oil and the insect control agent, when combined, may have a synergistic effect. For example, the insect control activity of 29% DEET may be achieved with 5% DEET when included in a combination of the present invention.

The compositions of the present invention may comprise, in admixture with a suitable carrier and optionally with a suitable surface active agent, two or more plant essential oil compounds and/or derivatives thereof, natural and/or synthetic, including racemic mixtures, enantiomers, diastereomers, hydrates, salts, solvates and metabolites, etc.

A suitable carrier may include any carrier in the art known for plant essential oils so long as the carrier does not adversely effect the compositions of the present invention. The term “carrier” as used herein means an inert or fluid material, which may be inorganic or organic and of synthetic or natural origin, with which the active compound is mixed or formulated to facilitate its application to the container or carton or other object to be treated, or its storage, transport and/or handling. In general, any of the materials customarily employed in formulating repellents, pesticides, herbicides, or fungicides, are suitable. The compositions of the present invention may be employed alone or in the form of mixtures with such solid and/or liquid dispersible carrier vehicles and/or other known compatible active agents such as other repellants, pesticides, or acaricides, nematicides, fungicides, bactericides, rodenticides, herbicides, fertilizers, growth-regulating agents, etc., if desired, or in the form of particular dosage preparations for specific application made therefrom, such as solutions, emulsions, suspensions, powders, pastes, and granules which are thus ready for use. The compositions of the present invention can be formulated or mixed with, if desired, conventional inert pesticide diluents or extenders of the type usable in conventional insect control agents, e.g. conventional dispersible carrier vehicles such as gases, solutions, emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, soluble powders, dusting agents, granules, foams, pastes, tablets, aerosols, natural and synthetic materials impregnated with active compounds, microcapsules, coating compositions for use on seeds, and formulations used with burning equipment, such as fumigating cartridges, fumigating cans and fumigating coils, as well as ULV cold mist and warm mist formulations, etc.

The compositions of the present invention may further comprise surface-active agents. Examples of surface-active agents, i.e., conventional carrier vehicle assistants, that may be employed with the present invention, comprise emulsifying agents, such as non-ionic and/or anionic emulsifying agents (e.g. polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, albumin hydrolyzates, etc. and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate, etc.); and/or dispersing agents such as lignin, sulfite waste liquors, methyl cellulose, etc.

The compositions of the present invention may be used to control insects by either treating a host directly, or treating an area in which the host will be located. For example, the host may be treated directly by using a cream or spray formulation, which may be applied externally or topically, e.g., to the skin of a human. A composition could be applied to the host, for example, in the case of a human, using formulations of a variety of personal products or cosmetics for use on the skin or hair. For example, any of the following could be used: fragrances, colorants, pigments, dyes, colognes, skin creams, skin lotions, deodorants, talcs, bath oils, soaps, shampoos, hair conditioners and styling agents.

In the case of an animal, human or non-human, the host may also be treated directly by using a formulation of a composition that is delivered orally. For example, a composition could be enclosed within a liquid capsule and ingested.

An area may be treated with a composition of the present invention, for example, by using a spray formulation, such as an aerosol or a pump spray, or a burning formulation, such as a candle or a piece of incense containing the composition. Of course, various treatment methods may be used without departing from the spirit and scope of the present invention. For example, compositions may be comprised in household products such as: air fresheners (including “heated” air fresheners in which insect repellent substances are released upon heating, e.g. electrically, or by burning); hard surface cleaners; or laundry products (e.g. laundry detergent-containing compositions, conditioners).

The present invention is further illustrated by the following specific but non-limiting examples. The following examples are prophetic, notwithstanding the numerical values, results and/or data referred to and contained in the examples. Examples 1 through 5 relate to the preparation of a cell line expressing the tyramine receptor (TyrR) and screening of compositions using this cell line. Examples 6 through 11 relate to the preparation of a cell line expressing the Or83b receptor, preparation of a cell line expressing the Or43a receptor, and screening of compositions using these cell lines. Examples 12 through 34 relate to the determination of the repellant effect and/or a pesticidal effect of compositions.

Example 1 Preparation of Stably Transfected Schneider Cell Lines with Tyramine Receptor (TyrR) A. PCR Amplification and Subcloning Drosophila Melanogaster Tyramine Receptor

Tyramine receptor is amplified from Drosophila melanogaster head cDNA phage library GH that is obtained through the Berkeley Drosophila Genome Project (Baumann, A., 1999, Drosophila melanogaster mRNA for octopamine receptor, splice variant 1B NCBI direct submission, Accession AJ007617). The nucleic acid sequence and the peptide sequence of TyrR are set forth in FIGS. 31A and 31B. Phage DNA is purified from this library using a liquid culture lysate. (Baxter, et al., 1999, Insect Biochem Mol Biol 29, 461-467). Briefly, oligonucleotides that are used to amplify the open reading frame of the Drosophila tyramine receptor (TyrR) (Han, et al., 1998, J Neurosci 18, 3650-3658; von Nickisch-Rosenegk, et al., 1996. Insect Biochem Mol Biol 26, 817-827) consist of the 5′ oligonucleotide: 5′ gccgaattgccaccATGCCATCGGCAGATCAGATCCTG 3′ and 3′ oligonucleotide: 5′ taatctagaTCAATTCAGGCCCAGAAGTCGCTTG 3′. Capitalized letters match the tyramine receptor sequence. An added Kozak sequence (Grosmaitre, X., Jacquin-Joly, E., 2001 Mamestra brassicae putative octopamine receptor (OAR) mRNA, complete cds. NCBI direct submission, Accession AF43878) is indicated by underlined nucleotides. The 5′ oligonucleotide also contains an EcoR I site and the 3′ oligonucleotide a Xba I site. The PCR is performed using Vent polymerase (New England Biolabs) with the following conditions: about 95° C., about 5 min for about 1 cycle; about 95° C., about 30 sec; and about 70° C., about 90 sec for about 40 cycles; and about 70° C., about 10 min for about 1 cycle.

The PCR product is digested with EcoR I and Xba I, subcloned into pcDNA 3 (Invitrogen) and sequenced on both strands by automated DNA sequencing (Vanderbilt Cancer Center). When this open reading frame is translated to protein, it is found to correctly match the published tyramine receptor sequence (Saudou, et al., The EMBO Journal vol 9 no 1, 6-617). For expression in Drosophila Schneider cells, the TyrR ORF is excised from pcDNA3 and inserted into pAC5.1/V5-His(B) [pAc5(B)] using the Eco RI and Xba I restriction sites.

For transfection, Drosophila Schneider cells are stably transfected with pAc5(B)-TyrR ORF using the calcium phosphate-DNA coprecipitation protocol as described by Invitrogen Drosophila Expression System (DES) manual. The precipitation protocol is the same for either transient or stable transfection except for the use of an antibiotic resistant plasmid for stable transfection. At least about ten clones of stably transfected cells are selected and separately propagated. Stable clones expressing the receptors are selected by whole cell binding/uptake using ³H-tyramine. For this assay, cells are washed and collected in insect saline (170 mM NaCl, 6 mM KCl, 2 mM NaHCO₃, 17 mM glucose, 6 mM NaH₂PO₄, 2 mM CaCl₂, and 4 mM MgCl₂). About 3 million cells in about 1 mL insect saline are incubated with about 4 nM ³H-tyramine at about 23° C. for about 5 minutes. Cells are centrifuged for about 30 seconds and the binding solution is aspirated. The cell pellets are washed with about 500 μL insect saline and the cells are resuspended and transferred to scintillation fluid. Nonspecific binding is determined by including about 50 μM unlabeled-tyramine in the reaction. Binding is quantified counting radioactivity using a using a Liquid Scintillation β-counter (Beckman, Model LS1801).

B. Selection of Clones Having the Highest Level of Functionally Active Tyramine Receptor Protein

Tyramine receptor binding/uptake is performed to determine which of the transfected clones have the highest levels of functionally active tyramine receptor protein. There are about 10 clonal lines for tyramine receptor and about 2 pAc(B) for control. ³H-tyramine (about 4 nM/reaction) is used as a tracer, with and without about 50 μM unlabeled tyramine as a specific competitor. For this assay, cells are grown in plates and are collected in about 3 ml of medium for cell counting and the number of cells is adjusted to about 3×10⁶ cells/ml. About two pAcB clones are used in parallel as controls. About 1 ml cell suspension is used per reaction. Based on specific binding, about 3 clones express a high level of active tyramine receptor protein. The clone having the highest specific tyramine receptor binding (about 90%), is selected for further studies. The selected clone is propagated and stored in liquid nitrogen. Aliquot of the selected clone are grown for whole cell binding and for plasma membrane preparation for kinetic and screening studies. The control pAcB does not demonstrate any specific binding for the tyramine receptor.

C. Efficacy of Schneider Cells Transfected with Tyramine Receptor for Screening Compositions for Tyramine Receptor Interaction

Cells transfected with the tyramine receptor (about 1×10⁶ cells/ml) are cultured in each well of a multi-well plate. About 24 hours after plating the cells, the medium is withdrawn and replaced with about 1 ml insect saline (about 23° C.). Different concentrations of ³H-tyramine (about 0.1-10 nM) are added with and without about 10 μM unlabeled tyramine and incubated at room temperature (RT). After about a 20 minute incubation, the reaction is stopped by rapid aspiration of the saline and at least one wash with about 2 ml insect saline (about 23° C.). Cells are solubilized in about 300 μl 0.3M NaOH for about 20 min at RT. Solubilized cells are transferred into about 4 ml Liquid Scintillation Solution (LSS) and vigorously vortexed for about 30 sec before counting the radioactivity using a Liquid Scintillation β-counter (Beckman, Model LS1801) (LSC).

With reference to FIG. 1, receptor specific binding data is expressed as fmol specific binding per 1×10⁶ cells and measured as a function of ³H-tyramine concentration. Specific binding values are calculated as the difference between values in the absence of and values in the presence of about 10 μM unlabeled tyramine. As shown in FIG. 1, the maximum specific binding occurs at about 5 nM ³H-tyramine. Untransfected cells do not respond to tyramine at concentration as high as about 100 μM.

To study the kinetics of the tyramine receptor in stably transfected cells with pAcB-TyrR, crude membrane fractions are prepared from the transfected cells and used to calculate the equilibrium dissociation constant (K_(d)), Maximum Binding Capacity (B_(max)), equilibrium inhibitor dissociation constant (K_(i)) and EC₅₀ (effective concentration at which binding is inhibited by 50%). A preliminary study to determine the optimum concentration of membrane protein for receptor binding activity is performed. In this study, different concentrations of protein (about 10-50 μg/reaction) are incubated in about 1 ml binding buffer (50 mM Tris, pH 7.4, 5 mM MgCl₂ and 2 mM ascorbic acid). The reaction is initiated by the addition of about 5 nM ³H-tyramine with and without about 10 μM unlabeled tyramine. After about 1 hr incubation at room temperature, reactions are terminated by filtration through GF/C filters (VWR), which have been previously soaked in about 0.3% polyethyleneimine (PEI). The filters are washed one time with about 4 ml ice cold Tris buffer and air dried before the retained radioactivity is measured using LSC. Binding data is analyzed by curve fitting (GraphPad software, Prism). The data demonstrates no differences between about 10, 20, 30 and 50 μg protein/reaction in tyramine receptor specific binding. Therefore, about 10 μg protein/reaction is used.

To determine B_(max) and K_(d) values for tyramine receptor (TyrR) in membranes expressing TyrR, saturation binding experiments are performed. Briefly, about 10 μg protein is incubated with ³H-tyramine at a range of concentrations (about 0.2-20 nM). Binding data is analyzed by curve fitting (GraphPad software, Prism) and the K_(d) for tyramine binding to its receptor is determined.

To determine the affinities of several ligands for TyrR, increasing concentration of several compounds are tested for their ability to inhibit binding of about 2 nM ³H-tyramine. For both saturation and inhibition assays total and non-specific binding is determined in the absence and presence of about 10 μM unlabeled-tyramine, respectively. Receptor binding reactions are incubated for about 1 hr at room temperature (RT) in restricted light. Reactions are terminated by filtration through GF/C filters (VWR), which have been previously soaked in about 0.3% polyethyleneimine (PEI). The filters are washed one time with about 4 ml ice cold Tris buffer and air dried before retained radioactivity is measured using LSC. Binding data is analyzed by curve fitting (GraphPad software, Prism).

With reference to FIG. 2, depicting a saturation binding curve of ³H-tyramine (³H-TA) to membranes prepared from Schneider cells expressing tyramine receptor, H-tyramine has a high affinity to tyramine receptor in the stably transfected cells with pAcB-TyrR with K_(d) determined to be about 1.257 nM and B_(max) determined to be about 0.679 pmol/mg protein.

With reference to FIG. 3, depicting the inhibition binding of ³H-tyramine (³H-TA) to membranes prepared from Schneider cells expressing tyramine receptor in the presence and absence of various concentrations of unlabeled tyramine (TA), the EC₅₀ and the K_(i) for tyramine against its receptor in Schneider cells expressing tyramine receptor are about 0.331 μM and 0.127 μM, respectively.

In order to determine the pharmacological profile of tyramine receptor (TyrR), the ability of a number of putative Drosophila neurotransmitters to displace ³H-tyramine (³H-TA) binding from membranes expressing tyramine receptor is tested. With reference to FIG. 4, depicting inhibition binding of ³H-Tyramine to membranes prepared from Schneider cells expressing tyramine receptor in the presence and absence of different concentrations of unlabeled ligands (including Tyramine (TA), Octopamine (OA), Dopamine (DA), and Serotonin (SE)), tyramine displays the highest affinity (K_(i) of about 0.127 μM, EC₅₀ of about 0.305 μM) for the Drosophila TyrR. Octopamine, dopamine and serotonin were less efficient than tyramine at displacing ³H-tyramine binding.

With reference to Table A, setting forth the K_(i) and EC₅₀ of the ligands, the rank order of potency is as follows: tyramine>octopamine>dopamine>serotonin, showing the likelihood that the stably transfected Schneider cells are expressing a functionally active tyramine receptor.

TABLE A Ligand K_(i) (μM) EC₅₀ (μM) Tyramine (TA) 0.127 0.305 Octopamine (OA) 2.868 7.456 Dopamine (DA) 5.747 14.940 Serotonin (SE) 8.945 23.260 As such, Schneider cells expressing tyramine receptor are effective as a model for studies and screening for compositions that interact with the tyramine receptor.

Example 2 Treatment of Cells Expressing the Tyramine Receptor and Effect of Compositions on Intracellular [cAMP]

Cells are grown on dishes and the media changed the day before the treatment. When cells are approximately 95% confluent, media is aspirated and the cells are washed one time with about 5 mL of about 27° C. insect saline (170 mM NaCl, 6.0 mM KCl, 2.0 mM NaHCO₃, 17.0 mM glucose, 6.0 mM NaH2PO4, 2.0 mM CaCl2, 4.0 mM MgCl2; pH 7.0). About 20 mL of insect saline is added, and cells are harvested by gentle scraping. An aliquot of the cells is counted by hemocytometer, and the cells are then centrifuged for about 5 minutes at about 1000 RPM. Cells are resuspended to give about 3×10⁶ cells per mL. IBMX is added to about 200 μM. Then about 1 mL of cell suspension is aliquoted for treatment. Forskolin (cAMP inducing agent), tyramine or different composition candidates are added, and the cells are incubated at about 27° C. for about 10 minutes.

Treated cells are centrifuged at about 13000 g for about 10 seconds. The solution is aspirated and about 1 mL of about −20° C. 70% ethanol is added. The cell pellet is disrupted by vortexing and the samples placed at about −20° C. overnight. Following the ethanol extraction, cellular debris is pelleted by centrifugation at about 13000 g for about 5 minutes. The supernatant is transferred to a tube and lyophilized to dryness in a rotary speed-vac. The resulting extract is resuspended in about 100 μL TE and used for the cAMP assay.

The cAMP assay is based on competition binding between endogenous cAMP and ³H-cAMP to a cAMP binding protein. The ³H-cAMP Biotrak system (Amersham Biosciences) is used for this assay as per the manufacturer's instructions. Briefly, about 50 μL of the cellular extract is incubated with about 50 μL ³H-cAMP and about 100 μL cAMP binding protein in an ice bath for about 2-4 hours. Charcoal (about 100 μL) is then added and the solution centrifuged for about 3 minutes at about 4° C. About 200 μL of the reaction mixture is removed and levels of ³H-cAMP are determined by scintillation counting. Levels of endogenous cAMP from the cells are calculated using a standard curve with cold cAMP ranging from about 0 to 16 pmol per reaction.

Example 3 Treatment of Cells Expressing the Tyramine Receptor and Effect of Compositions on Intracellular [Ca²⁺]

Intracellular calcium ion concentrations ([Ca²⁺]i) are measured by using the acetoxymethyl (AM) ester of the fluorescent indicator fura-2 (Enan, et al., Biochem. Pharmacol vol 51, 447-454). In this study, cells expressing tyramine receptor are grown under standard conditions. A cell suspension is prepared in assay buffer (140 mM NaCL, 10 mM HEPES, 10 mM glucose, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2) and cell number adjusted to about 2×10⁶ cells per ml. Briefly, about 1.0 ml cell suspension (about 2×10⁶ cells) is incubated with about 5 μM Fura 2/AM for about 30 min at about 28° C. After incubation, the cells are pelleted at about 3700 rpm for about 10 sec at room temperature and then resuspended in about 1.5 ml assay buffer. [Ca²⁺]i changes are analyzed in spectrofluorometer in the presence and absence of test chemicals. Excitation wave lengths are about 340 nm (generated by Ca²⁺-bound fura-2) and about 380 nm (corresponding to Ca²⁺-free fura-2). The fluorescence intensity is monitored at an emission wave length of about 510 nm. No absorbance of fluorescence artifacts are observed with any of the compounds used. The ratio of about 340/380 nm is calculated and plotted as a function of time.

Example 4 Effect of Lilac Flower Oil and Black Seed Oil on Tyramine Receptor Binding Activity in Cells Expressing the Tyramine Receptor

To determine whether specific oils, namely, Lilac Flower Oil (LFO) and Black seed Oil (BSO), interact and regulate the functional expression of tyramine receptor, membranes from stably transfected and untransfected Schneider cells are analyzed for ³H-Tyramine binding.

For the interaction with ³H-Tyramine at the receptor sites, the same binding protocol as described above is used. A dose-response of LFO and BSO (about 1-100 μg/ml) is performed to determine their effect on the inhibition binding of ³H-Tyramine to membranes prepared from Schneider cells expressing the tyramine receptor. With reference to FIG. 5, depicting the inhibition binding of ³H-Tyramine to membranes prepared from Schneider cells expressing tyramine receptor in the presence and absence of different concentrations of LFO and BSO, the inhibition of ³H-Tyramine to its receptor is demonstrated in response to treatment with LFO and BSO in a dose-dependent manner. The EC₅₀ values for LFO and BSO are approximately in the neighborhood of 10 μg/ml and 20 μg/ml, respectively.

Turning now to FIG. 6, depicting the inhibition binding of ³H-tyramine to membranes prepared from Schneider cells expressing tyramine receptor in the presence and absence of either LFO or BSO or in combination with about 1 and 10 μM unlabeled Tyramine, LFO (about 25 μg/ml) by itself inhibits the binding of ³H-Tyramine to its receptor. This effect is equivocal to the effect of about 10 μM (about 1.74 μg/ml) unlabeled tyramine. In addition, LFO potentiates the potency of unlabeled Tyramine against ³H-Tyramine binding only when unlabeled tyramine is used at about 1 μM. On the other hand, BSO (about 25 μg/ml) is less efficacious against ³H-Tyramine binding than LFO. BSO, however, significantly increases the potency of unlabeled-Tyramine against ³H-Tyramine binding regardless the concentration of unlabeled Tyramine. The two oils do not show any effect on ³H-Tyramine binding in untransfected Schneider cells.

As such, it appears that LFO and BSO interact with the tyramine receptor differently. Not wishing to be bound by theory or mechanism, it is likely that LFO and tyramine compete at the same binding sites, while BSO acts at different sites of the receptor than the endogenous ligand (tyramine). Certain other oils, including those expressly set forth in this application, also interact with the tyramine receptor.

Example 5 Effect of Lilac Flower Oil and Black Seed Oil on Intracellular [cAMP] in Cells Expressing the Tyramine Receptor

To examine test chemical-dependent coupling of the tyramine receptor, pAcB-TyrR is stably expressed in Schneider cells. Transfected and untransfected cells are treated with tyramine (about 10 μM), LFO (about 25 μg/ml) and BSO (about 25 μg/ml) in the presence and absence of forskolin (FK) (about 10 μM). cAMP production is measured using the ³H-cAMP assay kit (Amersham) as described above.

To ensure that the cAMP cascade in this cell model is functionally active, forskolin, a cAMP inducer, is used as standard agent. As shown in FIGS. 7 through 9, which depict tyramine-dependent changes in cAMP levels in Schneider cells expressing tyramine receptor following treatment with LFO (about 25 μg/ml) and BSO (about 25 μg/ml) in the presence and absence of tyramine (about 10 μM) and forskolin (about 10 μM), there is about a 19-fold increase in the cAMP levels only in transfected cells in response to treatment with forskolin as compared to the basal level of cAMP in cells treated only with the solvent (ethanol).

Tyramine, on the other hand, induces a slight decrease (about 10%) in cAMP production. Tyramine, however, significantly antagonizes forskolin-stimulated cAMP levels in cells expressing tyramine receptor, suggesting that tyramine receptor couples to G_(αi/o) in the presence of tyramine, as shown in FIG. 7. About a 34% and 25% decrease in cAMP level are found only in transfected cells in response to treatment with LFO and BSO respectively (FIG. 8). While tyramine potentiates the effect of LFO on cAMP production in the tyramine-receptor transfected cells, co-treatment of BSO and tyramine does not induce any changes in cAMP level beyond the effect of BSO by itself, as shown in FIG. 8. The LFO- and BSO-decreased cAMP levels in Schneider cells expressing tyramine receptor is diminished in the presence of forskolin, as shown in FIG. 9.

Treatment with certain other plant essential oils, including those expressly set forth in the application, also result in changes in intracellular cAMP levels in cells expressing tyramine receptor.

Example 6 Preparation of Stably Transfected Schneider Cell Lines with Olfactory receptors (Or83b and Or43a)

A. RT-PCR Amplification and Subcloning Drosophila Melanogaster Olfactory Receptors Or83b and Or43a

Total RNA is prepared from the head and antenna of wild type Drosophila melanogaster using Trizol Reagent (Invitrogen). They are homogenized in the Trizol using a motor driven teflon pestle and glass homogenizer. RNA is then prepared as per the manufacturer's instructions and includes removal of proteoglycans and polysaccharides by precipitation. The total RNA is reverse transcribed using oligo-dT as a primer and MuLV reverse transcriptase (Applied Biosystems). To PCR amplify the open reading frames, the following oligonucleotides are used: Or83b Sense 5′ taagcggccgcATGACAACCTCGATGCAGCCGAG 3′; Or83b Antisense 5′ ataccgcggCTTGAGCTGCACCAGCACCATAAAG 3′; Or43a Sense 5′ taagcggccgcATGACAATCGAGGATATCGGCCTGG 3′; and Or43a Antisense 5′ ataccgcggTTTGCCGGTGACGCCACGCAGCATGG 3′. Capitalized letters match the Or83b and Or43a receptors sequence. The Sense oligonucleotides contain Not I sites and the antisense oligonucleotides contain Sac II sites. Both restriction sites are indicated by underlined nucleotide. The antisense oligonucleotides do not contain stop codons so the V5 epitope from the pAC 5.1 plasmid will be in frame with the translated proteins. For PCR amplification of Or83b, Vent polymerase (New England Biolabs) is used with the following conditions: about 95° C., about 5 min for about 1 cycle; about 95° C., about 30 sec; and about 70° C., about 90 sec for about 40 cycles; and about 70° C., about 10 min for about 1 cycle. For PCR amplification of Or43a, the Failsafe PCR premix selection kit (Epicentre Technologies) is used with the following conditions: about 95° C., about 5 min for about 1 cycle; about 95° C., about 30 sec; about 60° C., about 30 sec and about 70° C., about 90 sec for about 50 cycles; and about 70° C., about 10 min for about 1 cycle. The Failsafe premix buffer F yields the correctly sized product. The PCR products are digested with Sac II and Not I, gel purified and ligated into pAC 5.1/V5 His B (Invitrogen). Inserts are sequenced on both strands by automated flourescent sequencing (Vanderbilt Cancer Center). Both the Or83b open reading frame and Or43a open reading frame code for identical proteins as compared to sequence information on PubMed and found in the genomic sequence on the Web site. The nucleic acid sequence and the peptide sequence of Or43a are set forth in FIGS. 32A and 32B. The nucleic acid sequence and the peptide sequence of Or83b are set forth in FIGS. 33A and 33B.

For transfection, Drosophila Schneider cells are stably transfected with pAc5(B)-Or83b ORF or pAc5(B)-Or43a ORF using the calcium phosphate-DNA coprecipitation protocol as described by Invitrogen Drosophila Expression System (DES) manual as described above. At least about ten clones of stably transfected cells with either Or83b or Or43a are selected and separately propagated. Stable clones are analyzed to test whether they express corresponding mRNA using RT-PCR method. RNA is prepared from cells using Trizol as per the manufacturer's instructions. Total RNA is reverse transcribed with MuLV Reverse Transcriptase. PCR is performed using Vent polymerase and the following primers: Or83b sense and Or83b antisense; Or43a sense and Or43a antisense. PCR products are analyzed by agarose gel electrophoresis and compared to control Schneider cell RNA used for RT-PCR. A clone that highly expresses Or83b-mRNA or Or43a-mRNA is used in further studies to address protein expression (Western blot), and signaling (cAMP production and [Ca2+]) in response to treatment with tyramine and certain plant essential oils.

RT-PCR is used to determine which clones expressed the Or83b and Or43a genes. Agarose gel analysis indicates that for Or83b, about 4 clones out of about 10 clones yield the correct size product of about 1.46 kb. Likewise, for Or43a, about 2 clones yield the correct size product of about 1.1 kb. Neither of these products is obtained when PCR is performed on the control Schneider cells. Clones expressing the mRNA are chosen for additional studies with the receptor.

B. Efficacy of Schneider Cell Lines Transfected with Or83b Receptor or Or43a Receptor for Screening Compositions for Or83b and Or43a Receptor Interaction

To address whether Or83b receptor and Or43a receptor contain a specific binding site for tyramine, membranes expressing Or83b receptor or Or43a receptor are prepared from cells expressing either receptor, as described above, and used for competitive binding with ³H-tyramine. The binding assay protocol is exactly as described for cells expressing TyrR, as described above. As shown in FIG. 10, depicting a saturation binding curve of ³H-tyramine to membranes prepared from Schneider cells expressing the Or83b receptor in the presence or absence of about 20 μM unlabeled tyramine, and FIG. 11, depicting the same information for the cells expressing the Or43a receptor, ³H-Tyramine binds specifically to the Or83b and the Or43a receptors. As set forth in Table B, Tyramine binds to the Or83b receptor with Kd of approximately 96.90 nM and B_(max) of approximately 4.908 pmol/mg protein. For Or43a the corresponding values are Kd of approximately 13.530 nM and Bmax of approximately 1.122 pmol/mg protein.

TABLE B Receptor type K_(i) (nM) B_(max) (pmol/mg protein) TyrR 1.257 0.679 Or83b 96.900 4.908 Or43a 13.530 1.122

Example 7 Production of cAMP in Cells Expressing Olfactory Receptors

To ensure that the cAMP cascade in this cell model is functionally active, forskolin, a cAMP inducer, is used as standard agent. Cyclic-AMP levels are measured using the cAMP assay described above in Example 2. As shown in FIG. 12, depicting forskolin-dependent changes in cAMP levels in the cells expressing Or83b receptor, there is approximately a 13-fold increase from the basel cAMP levels in cells treated with about 10 μM forskolin for about 10 min at room temperature. Similar results are obtained with cells expressing Or43a receptor. As such, the cells expressing olfactory receptors have a functionally active cAMP cascade.

Example 8 Intracellular mobilization of Ca²⁺ in Cells Expressing Olfactory Receptors

Intracellular Ca²⁺ levels are measured using the method described above in Example 3. Calcium mobilization occurs in cells expressing either Or83b or Or43a receptor in response to treatment with ionomycin (a Ca²⁺ inducing agent) and tyramine. Specifically, with reference to FIGS. 13 and 14, in which fluorescence ratio determined from excitation with 340 nm and 380 nm correlates to intracellular calcium levels when about 2 μM ionomycin is added to the Or83b or Or43a expressing cells, a marked increase in intracellular calcium is detected.

Approximately 3.8-fold and 7-fold increases in calcium are found in cells expressing Or83b and Or43a, respectively, in response to treatment with ionomycin. With reference to FIG. 15, testing of the tyramine at about 10 μM can also induce approximately a 1.18-fold increase and 3.5-fold increase in intracellular calcium in cells expressing Or83b and Or43a, respectively.

Collectively, the pharmacological analysis data confirm that these cell models that were transfected with either Or83b receptor gene or Or43a receptor gene are expressing functioning protein receptors.

Example 9 Effect of Various Plant Essential Oils on the Binding Activity of Olfactory Receptors and Signaling Pathways Down Stream to the Receptors

The cells expressing one of the olfactory receptors are used to investigate the interaction of plant essential oils with these receptors and the signaling cascade downstream of each receptor.

For the binding activity, membranes are prepared from each cell model and used to investigate the interaction of plant essential oil with the receptor binding site. With reference to FIG. 16, the following oils interact with the olfactory receptors: lilac flower oil (LFO), diethyl phthalate, α-terpineol, and piperonal.

Likewise, with reference to FIGS. 17 and 18, the following oils interact with the olfactory receptors: black seed oil (BSO), α-pinene, quinone, p-cymene, sabinene, α-thujone and d-limonene.

Similarly, with reference to FIGS. 19 through 21, the following oils also interact with the olfactory receptors: geraniol, linalyl anthranilate, phenyl acetaldehyde, linalool, α-terpineol, t-anethole, terpinene 900, lindenol, eugenol, thyme oil, carvacrol, thymol, piperonal, piperonyl alcohol, piperonyl acetate, and piperonyl amine.

Certain other oils, including those expressly set forth in this application, also interact with the olfactory receptors.

Example 10

Effect of Various Plant Essential Oils on Intracellular Mobalization of Ca²⁺ in Cells Expressing the Or43a Receptor

To determine the effect of various plant essential oils on intracellular calcium mobilization, intact cells from each cell model are used in the assay as described above. Changes in intracellular Ca²⁺ levels are calculated based on the difference between the 340/380 fluorescence ratios before and after approximately 150 seconds of the treatment. As shown in FIG. 22, treatment with ionomycin and tyramine, which induce mobilization of Ca²⁺ in control cells, increases the intracellular Ca²⁺ levels only negligibly in cells expressing the Or43a receptor.

With reference to FIGS. 22 through 28, the following oils result in calcium mobilization in cells expressing the Or43a receptor: linalyl anthranilate, linalool, perillyl alcohol, t-anethole, geraniol, phenyl acetaldehyde, eugenol, piperonyl alcohol, piperonyl acetate, piperonyl amine, α-terpineol, lindenol, terpinene 900, thyme oil, tmymol, carvacrol, LFO, BSO, α-pinene, p-cymene, d-limonene, sabinen, quinine, l-carvone, d-carvone, and α-thujone. Finally, as shown in FIG. 24, treatment of piperonal decreases the intracellular Ca²⁺ levels in cells expressing the Or43a receptor.

Treatment with certain other plant essential oils, including those expressly set forth in the application, also cause changes in intracellular Ca²⁺ levels in cells expressing the Or43a receptor.

Additionally, treatment with certain other plant essential oils, including those expressly set forth in the application, cause changes in intracellular Ca²⁺ levels in cells expressing the Or83b receptor.

Example 11 Effect of Various Plant Essential Oils on Camp Production in Cells Expressing Olfactory Receptors

To determine the effect of various plant essential oils on intracellular cAMP production and the tyramine-dependent changes of cAMP in cells expressing one of the olfactory receptors, cells from each cell model are treated with LFO (about 50 μg/ml) and BSO (about 50 μg/ml) in the presence and absence of tyramine (about 20 μM) and forskolin (about 10 μM) and intracellular cAMP is thereafter qualified using the cAMP assay described above in Example 2.

As shown in FIGS. 29 and 30, treatment with the following oils result in an increase in cAMP levels in cells expressing Or43a receptor: tyramine; LFO; BSO; LFO and tyramine; BSO and tyramine; forskolin; tyramine and forskolin; LFO and forskolin; LFO, forskolin and tyramine; BSO; and BSO, tyramine and forskolin.

Still referring to FIGS. 29 and 30, approximately 34%, 32% and 64% increases in cAMP production in cells expressing Or83b receptor are produced in response to treatment with about 20 μM tyramine, about 50 μg LFO/ml and about 50 μg BSO/ml, respectively. An antagonistic effect (about 24%) on cAMP production is found in response to co-treatment with tyramine and LFO, as compared to the effect of each one by itself. On the other hand, a synergistic effect (about 300% increases) of cAMP production is found in response to co-treatment with BSO and tyramine.

In the presence of forskolin (about 10 μM), approximately a 3000-fold increase in the production of cAMP is found. When forskolin-pretreated cells administered with either tyramine or LFO, only approximately a 10-13% increase of cAMP production is found beyond the effect of forskolin by itself. The addition of BSO to forskolin-pretreated cells induces about 22% more increase in the cAMP levels beyond the forskolin-induced cAMP production in these cells.

Additionally, treatment with certain other plant essential oils, including those expressly set forth in this application, result in changes in the intracellular cAMP levels in cells expressing either the Or43a or the Or83b receptor.

Example 12 Toxicity of Compositions on Drosophila Melanogaster Fly

Two acetonic solutions (about 1% and 10%) from a test composition are prepared. Test concentration in acetone are then added to the inside of glass vials (about 5 mL) that are marked to about 3 cm above the bottom. The vials are rotated such that the inner surfaces of the vials, except the area between the marks to the neck, are left with a film of test composition. All vials are aerated for about 10 sec to ensure complete evaporation of acetone before introducing the flies to the treated vials. After complete evaporation of acetone, about 10 adult sex mixed flies are added to each vial and the vials are stoppered with cotton plugs. Mortality is observed about 24 hr after exposure.

Example 13 Toxicity of Lilac Flower Oil (LFO) and Black Seed Oil (BSO) on Wild-Type Fruit Fly and Tyramine-Receptor Mutant Fly

Wild-type Drosophila Melanogaster (fruit fly) and tyramine-receptor mutant fruit fly are used as a model to determine the toxicity of LFO and BSO. The toxicity of these oils is studied using the method described above in Example 12. With reference to Tables C and D below, both chemicals are toxic to wild type fruit flies. LFO is about 300-fold more toxic to the fruit flies than BSO. The LC₅₀ for LFO is in the neighborhood of about 25-30 ng/mm² and the corresponding value for BSO is about 94 μg/cm². On the other hand, LFO is at least about 1000-fold less toxic against the tyramine receptor mutant fly than BSO. The toxicity of both chemicals against the fruit fly is mediated through the tyramine receptor. While the mutation of tyramine receptor significantly reduces LFO toxicity against the fruit fly, the same mutation develops a more susceptible strain of fruit fly to BSO.

TABLE C Tyramine receptor [LFO] Wild/type flies [LFO] mutant flies ng/cm² Dead/alive % mortality μg/cm² Dead/alive % mortality 10  0/30 0.00 20  0/30 0.00 15  8/30 26.66 24  0/30 0.00 20 10/30 33.33 26  5/30 16.66 25 13/30 43.33 30 11/30 36.66 30 18/30 60.00 35 22/30 73.33 35 25/30 83.33 38 28/30 93.33 40 30/30 100.00 40 30/30 100.00

TABLE D Tyramine receptor [BSO] Wild/type flies [BSO] mutant flies μg/cm² Dead/alive % mortality μg/cm² Dead/alive % mortality 18.90  0/30 00.00 18.90  5/20 25 37.74  3/30 10.00 37.74  8/20 40 56.60  8/30 26.66 56.60 15/20 75 94.34 15/30 50.00 94.34 18/20 90 141.51 21/30 70.00 141.51 20/20 100 188.68 30/30 100.00

Example 14 Repellent Effect of Compositions on Farm Ants

Adult insect are randomly selected for testing the repellent effect of compositions and are not individually marked. About 5 insects per replicate are used. About 3 replicates are used for each treatment. Untreated control tests are included with only solvent (acetone) application to an equal sized population/replications, held under identical conditions. A filter paper (about 80 cm²) is treated with the composition (about 100 mg in 300 ml acetone). After about 3 min of air drying, the filter paper is placed in a dish and repellency against insects is performed. Insects are released to the dish, one insect at a time at the far end of the dish. Using one or more stopwatches, the time spent on either the filter paper or the untreated surface of the dish is recorded up to about 300 seconds. Repellency ratio (RR) is calculated as follows: RR=[(time on control surface−time on treated surface)/total time of test]. If RR>0 then the composition is considered to have a repellant effect, that is to say, an effect, wherein more insects are repelled away from treated surface than the control surface; if RR<0 then the composition is considered not to have a repellant effect.

Example 15 Repellent Effect of Lilac Flower Oil (LFO) and Black Seed Oil (BSO) on Farm Ants

The repellent effect of LFO (about 1.4 mg/cm²) and BSO (about 1.4 mg/cm²) against farm ants is studied using the method described above in Example 14. As shown in Tables E and F, BSO demonstrates more repellency against farm ants than LFO. Approximately 90% and 100% repellency against farm ants is provided by LFO and BSO, respectively. Additionally, LFO and BSO also induce 100% mortality against farm ants within 24 hr of exposure.

TABLE E Replicate Time on LFO test surface (sec) number Treated surface Untreated surface Repellency % R1 26.4 273.6 82.4 R2 10.8 289.2 92.8 R3  9.4 290.6 93.7 X ± SD 15.53 ± 7.7 284.47 ± 7.7 89.63 ± 5.1

TABLE F Replicate Time on BSO test surface (sec) number Treated surface Untreated surface Repellency % R1 0 300 100 R2 0 300 100 R3 0 300 100 X ± SD 0 ± 0 300 ± 0 100 ± 0

A dish treated with BSO is also used to address the residual effect of BSO on repellency against ants. Five ants are used per day according to the repellency protocol described above. In parallel, time-course toxicity for BSO is determined. In the toxicity experiment, an ant is exposed to the same treated surface for about 10 sec, and then transferred to a fresh container. Mortality data is recorded about 24 hr after exposure. Five ants are used per day. As shown in Table G, BSO provides repellency against farm ants up to about 4 days.

TABLE G Time elapsed after surface treatment, days Repellency % Day 1 100 Day 2 100 Day 3 100 Day 4 100

Example 16 Repellent Effect of d-Limonene, α-Pinene, and p-Cymene, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

With reference to Table H, d-limonene, α-pinene, and p-cymene each demonstrate repellency alone. However, when the oils are mixed to form Composition A, a composition including about one third each of d-limonene, α-pinene and p-cymene, there is a synergistic effect and the percent repellency is greatly increased.

TABLE H Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 α-pinene 77.4 48.4 139.2 07.2 p-cymene 86.2 42.5 133.6 10.9 Composition A 0.2 99.9 0.0 100.0 0.0 100 NO

Likewise, and with reference to Table I, d-limonene and α-pinene each demonstrate repellency alone. However, when the oils are mixed to form Composition B, a composition including about half each d-limonene and α-pinene, there is a synergistic effect and the percent repellency is greatly increased.

TABLE I Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 α-pinene 77.4 48.4 139.2 07.2 Composition B 1.0 99.3 1.0 99.3 NO

Example 17 Repellent Effect of Linalool, d-Limonene, α-Pinene, p-Cymene and Thyme Oil, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table J, although d-limonene, α-pinene, p-cymene and thyme oil each display repellency, Composition C, a composition including about 25% of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE J Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 α-pinene 77.4 48.4 139.2 07.2 p-cymene 86.2 42.5 133.6 10.9 thyme oil 58.0 61.3 Composition C 0.4 99.7 3.0 98.0 1.8 98.8 2.4 98.4

Likewise, as shown in Table K, although linalool, α-pinene, p-cymene and thyme oil each display repellency, Composition D, a composition including about 25% of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE K Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % linalool 59.0 60.7 111.2 25.9 α-pinene 77.4 48.4 139.2 07.2 p-cymene 86.2 42.5 133.6 10.9 thyme oil 58.0 61.3 Composition D 8.2 97.3 3.0 98.0

Similarly, as shown in Table L, although linalool, α-pinene, and p-cymene each display repellency, Composition E, a composition including about one third of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE L Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % linalool 59.0 60.7 111.2 25.9 α-pinene 77.4 48.4 139.2 07.2 p-cymene 86.2 42.5 133.6 10.9 Composition E 12.8 95.7 0.2 99.9 1.3 99.1 3.8 97.5

Example 18 Repellent Effect of α-Pinene, Thyme Oil, α-Thujone, Sabinene, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

Although α-pinene, thyme oil, α-thujone, and sabinene each display repellency, as shown in Table M, Composition F, a composition including about 25% of each of the oils, demonstrates enhanced repellency.

TABLE M Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % α-pinene 77.4 48.4 139.2 07.2 thyme oil 58.0 61.3 Composition F 3.2 98.9 0.0 100.0 0.0 100.0 0.0 100.0

Example 19

Repellent Effect of d-Limonene, p-Cymene, Thymol, Carvacrol and Geraniol, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table N, although d-limonene, p-cymene, thymol and carvacrol each display repellency, Composition G, a composition including about 25% of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE N Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 p-cymene 86.2 42.5 133.6 10.9 thymol 62.6 58.3 104.4 30.4 carvacrol ND NO Composition G 2.5 99.2 7.6 94.9 0.0 100.0 4.0 94.0

Likewise, as shown in Table O, although d-limonene, p-cymene, and thymol each display repellency, Composition H, a composition including about one third of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE O Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 p-cymene 86.2 42.5 133.6 10.9 thymol 62.6 58.3 104.4 30.4 Composition H 0.83 99.7 9.8 93.5 6.0 96 1.3 99.1

Similarly, as shown in Table P, although d-limonene, p-cymene, thymol, and geraniol each display repellency, Composition I, a composition including about 25% of each of the oils, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE P Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.2 27.6 p-cymene 86.2 42.5 133.6 10.9 thymol 62.6 58.3 104.4 30.4 geraniol 69 54.0 129.0 14.0 Composition I 1.6 98.7 0.2 99.9 6.3 95.8 4.25 97.2

Example 20 Repellent Effect of Linalyl Anthranilate, α-Pinene, d-Limonene, p-Cymene, and Geraniol, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table Q, although geraniol, d-limonene, p-cymene and linalyl anthranilate each display repellency, Composition J, a composition including about 40% geraniol, about 30% d-limonene, about 10% p-cymene, about 10% α-pinene and about 10% linalyl anthranilate, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE Q Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % geraniol 69.0 54.0 129.0 14.0 d-limonene 55.7 62.9 136.2 10.9 α-pinene 77.4 48.4 139.2 07.2 p-cymene 86.2 42.5 133.6 10.9 linalyl anthranilate 46.2 69.2 104.6 30.7 Composition J 0.0 100 0.0 100 0.2 99.9 0.0 100

Example 21 Repellent Effect of d-Limonene, Thymol, α-Terpineol, Piperonyl Acetate, Piperonyl Amine, and Piperonal, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table R, although d-limonene, thymol, α-terpineol, piperonyl acetate, piperonyl amine and piperonal each display repellency, Composition K, a composition including about 20% d-limonene, about 30% thymol, about 20% α-terpineol, about 10% piperonyl acetate, about 10% piperonyl amine and about 10% piperonal, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE R Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % d-limonene 55.7 62.9 136.4 75.9 NO thymol 62.0 58.3 104.4 30.4 α-terpineol 109.6 26.9 piperonylacetate 52.4 65.1 106.6 28.9 piperonylamine 77.6 48.3 111.2 25.9 piperonal 93.6 37.6 125.8 16.1 Composition K 0.0 100 1.2 99.4 1.2 99.4 0.3 99.8

Example 22 Repellent Effect of Geraniol, d-Limonene, Eugenol, Lindenol and Phenylacetaldehyde, Alone and in Combination, on Farm Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table S, although geraniol, d-limonene, eugenol, lindenol, and phenylacetaldehyde each display repellency, Composition L, a composition including about 50% geraniol, about 20% d-limonene, about 10% eugenol, about 10% lindenol, and about 10% phenylacetaldehyde, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE S Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % geraniol 69.0 54.0 129.4 14.0 d-limonene 55.7 62.9 133.6 10.9 eugenol 76.8 48.8 139.0 07.3 lindenol 144.2 04.0 phenyl-acetaldehyde 144.8 03.5 Composition L 0.0 100 0.0 100 0.2 99.9 0.0 100

Example 23 Repellent Effect Geraniol, Lemon Grass Oil, Eugenol and Mineral Oil, Alone and in Combination, on Carpenter Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table T, although geraniol, lemon grass oil and eugenol, each display repellency, Composition M, a composition including about 50% geraniol, about 40% lemon grass oil, and about 10% eugenol, demonstrates repellency which exceed that of any of its component oils being used alone. Geraniol, lemon grass oil and eugenol are all generally regarded as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA), and, as such, are exempt from EPA pesticide registration requirements.

TABLE T Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % Geraniol 69.0 129.0 129.0 14.0 Lemongrass oil 47.0 68.7 79.8 46.8 eugenol 76.8 48.8 139.0 7.3 Composition M 0.6 99.6 0.6 99.6 1.0 99.3 1.2 99.4

Likewise, as shown in Table U, although geraniol and lemon grass oil each display repellency, Composition N, a composition including about 70% geraniol and about 30% lemon grass oil, demonstrates repellency which exceed that of any of its component oils being used alone.

TABLE U Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % Geraniol 69.0 54.0 129.0 14.0 Lemongrass oil 47.0 68.7 79.8 46.8 Composition N 0.67 99.6 0.80 99.5

Additionally, as shown in Table V, the addition of mineral oil, to form Composition O, a composition including about 60% geraniol, about 30% lemon grass oil, and about 10% mineral oil, does not effect the synergism of geraniol and lemongrass oil. Mineral oil alone does not demonstrate repellency, but serves to stabilize the composition, limiting the evaporation of the active components. Mineral oil, like geraniol and lemongrass oil, is a GRAS compound.

TABLE V Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % Geraniol 69.0 54.0 129.0 14.0 Lemongrass oil 47.0 68.7 79.8 46.8 Mineral oil NO Composition O 0.33 99.8 2.2 98.5 3.0 98.0

Example 24 Repellent Effect Geraniol, Thymol, Lemon Grass Oil and Mineral Oil, Alone and in Combination, on Carpenter Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table W, although geraniol, thymol and lemon grass oil, each display repellency, Composition P, a composition including about 50% geraniol, about 20% thymol, about 20% lemon grass oil, and about 10% mineral oil, demonstrates repellency which exceed that of any of its component oils being used alone. Geraniol, thymol, lemon grass oil, eugenol and mineral oil are all generally regarded as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA), and, as such, are exempt from EPA pesticide registration requirements.

TABLE W Repellency % Day 0 Day 1 Day 2 Day 3 sec. on T sec. on T sec. on T sec. on T Test chemical surface R % surface R % surface R % surface R % Geraniol 69.0 54.0 129.0 14.0 thymol 62.0 58.3 104.4 30.4 lemongrass oil 47.0 68.7 79.8 46.8 mineral oil NO Composition P 0.0 100 0.0 100 0.2 99.9 3.8 97.5

Example 25 Repellent Effect Black Seed Oil (BSO), Lilac Flower Oil (LFO), Geraniol, Thymol, Lemon Grass Oil and Mineral Oil, Alone and in Combination, on Carpenter Ants

The repellent effect of various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Table X, geraniol, thymol and thyme oil, each display repellency. As shown in Table Y, Compositions Q through V, containing various combinations of a BSO, LFO, geraniol, thymol, thyme oil, mineral oil, safflower oil and castor oil, demonstrate enhanced repellency.

TABLE X Day 0 Test chemical sec. on T surface Repellency % geraniol 69 54.0 thymol 62 58.3 thyme oil 58 61.3 mineral oil NO safflower oil NO castor oil NO

TABLE Y Day 0 sec. on T Test chemicals surface Repellency % Composition Q 0.2 99.9 about 25% geraniol and about 75% BSO Composition R 1.0 99.3 about 25% geraniol, about 50% BSO, and about 25% mineral oil Composition S 1.0 99.3 about 25% geraniol, about 50% BSO, and about 25% safflower oil Composition T 1.6 98.9 about 25% geraniol, about 25% thymol, and about 50% BSO Composition U 2.3 98.5 about 25% thyme oil, about 50% BSO, and about 25% castor oil Composition V 0.4 99.7 about 50% geraniol and about 50% LFO

Example 26 Repellent Effect of Commercial Repellent 29% DEET on Carpenter Ants

For purposes of comparison to the repellent effect of various compositions made of various plant essential oils, the repellency of an insect control agent, the commercial repellent 29% DEET, which may be purchased under the name, REPEL® (Wisconsin Pharmacal Company, Inc, Jackson, Wyo.), is measured against Carpenter ants by treating a filter paper with the 29% DEET. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. As shown in Table Z, 29% DEET has a percent repellency at day 0 of about 98.4 percent. The percent repellency of LFO, BSO, and the compositions of the present invention are comparable, and in some cases higher, than the percent repellency of 29% DEET.

TABLE Z Repellency % Day 0 sec. on Test chemical T surface R % DEET 29% 02.4 98.4

Example 27 Repellent Effect of Commercial Repellent DEET, Alone and in Combination with Geraniol, Thymol, and Lemon Grass Oil or Geraniol, d-Limonene, Eugenol, Lindenol, and Phyenylacetaldehyde, on Carpenter Ants

The repellent effect of commercial repellent DEET and various plant essential oils is tested by treating a filter paper with the test oils. After about five minutes at room temperature, the paper is placed in a dish and ants are introduced one at a time. The repellency is determined as described above, in Example 14. Oils are tested alone. Additionally, oils are mixed to form compositions, which are then tested.

As shown in Tables AA and BB, treatment with DEET in concentrations of about 5 to 10% displays no signs of repellency. However, as shown in Table AA, when combined with Composition W, a composition comprising about 25% geraniol, 10% thymol, 10% lemon grass oil and mineral oil (from 45 to 55% depending on the final concentration of DEET), percent repellency approaches 100. Likewise, as shown in Table BB, when combined with Composition X, a composition comprising about 25% geraniol, 10% d-limonene, 5% eugenol, 5% lindenol, 5% phenylacetaldehyde and mineral oil (from 40 to 50% depending on the final concentration of DEET), percent repellency is approximately 97 to 98 percent. Also, as shown in Tables AA and BB, enhanced repellency is shown when the various oils are combined with DEET.

TABLE AA % Repellency Day 0 Day 1 Chemicals Sec on T % Repellency Sec on T % Repellency  5% DEET 282 (10)  NO 10% DEET 260 (6)  NO Composition W 50 (6)  67% 174 (6)  NO  5% DEET 2.6 (1.9) 98% 10 (2)  93% plus Composition W 10% DEET 0.2 (0.4) 99% 2.4 (1.8) 98% plus Composition W

TABLE BB % Repellency Day 0 Day 1 Chemicals Sec on T % Repellency Sec on T % Repellency  5% DEET 282 (10)  NO 10% DEET 260 (6)  NO Composition X 40 (5)  74% 145 (10)  2  5% DEET 4 (2) 97% 8.8 (4.0) 94% plus Composition X 10% DEET 2.6 (2.0) 98% 7.2 (4.1) 95% plus Composition X

Example 28 Pesticidal Effect of Compositions on Head Lice

Live adult head lice Pediculus humanus capitus are collected from female and male children between the age of about 4 and 11 living in the Karmos area, Alexandria, Egypt. The insects are collected using fine-toothed louse detector comb and pooled together. The collected lice are kept in dishes and used in the studies within about 30 minutes of their collection.

Various concentrations of the compositions being tested are prepared in water To allow the pesticidal effect of these compositions to be compared to that of a commercially available lice-killing agent, ivermectin, is dissolved in water. About 1 ml of each concentration of the compositions are applied to a dish, about 1 ml of the ivermectin solution is applied to a dish, and about 1 ml of water is applied to a control dish. About 10 adult head lice are introduced to each dish.

Treated and control dishes are kept under continuous observation and LT₁₀₀ is observed. LT refers to the time required to kill a given percentage of insects; thus, LT₁₀₀ refers to the time required to kill 100% of the lice. Head lice is considered dead if no response to a hard object is found.

Example 29 Pesticidal Effect of Compositions Including Geraniol, d-Limonene, Benzyl Alcohol, P-Cymene, and Lilac Flower Oil on Head Lice

The pesticidal effect of Composition Y, a composition including about 20% p-cymene, about 40% Lilac Flower Oil (LFO), about 30% benzyl alcohol, and about 10% mineral oil are studied using the method described above in Example 28. The LT₁₀₀ of this composition is compared to that of a commercially available lice-killing agent, ivermectin. As shown in Table CC, the lice treated with Composition Y are all killed more quickly than the lice treated with ivermectin.

TABLE CC Treatment LT₁₀₀ (minutes) Composition Y 3 Ivermectin 5

Example 30 Repellent Effect of Compositions to Mosquitoes A. Oral Delivery

Hairless or shaved mice and guinea pigs are used to test the repellent effect of compositions delivered orally. The test oil (e.g., lilac flower oil (LFO) or black seed oil (BSO)) or test composition (e.g., a composition containing geraniol, d-linonene, eugenol, and lindenol) is administered orally to about 10 rodents. A control substance, such as mineral oil, is administered orally to about 10 rodents. After approximately 30 minutes, each rodent is placed in an enclosed container. About 20 mosquitoes are introduced to each container. Each container is observed for approximately 1 hour. The time that each insect spends on the rodent is recorded and number of lesions caused by the insect on the skin of the rodent is recorded. The insects spend less time on rodents receiving the test compositions than on the rodents receiving the control substance. The rodents receiving the test compositions receive fewer lesions than the rodents receiving the control substances.

B. Topical Delivery

Hairless or shaved mice and guinea pigs are used to test the repellent effect of compositions delivered topically. The test oil (e.g., lilac flower oil (LFO) or black seed oil (BSO)) or test composition (e.g., a composition containing geraniol, d-linonene, eugenol, and lindenol) is administered topically to the skin of about 10 rodents. A control substance, such as mineral oil, is administered topically to the skin of about 10 rodents. After approximately 30 minutes, each rodent is placed in an enclosed container. About 20 mosquitoes are introduced to each container. Each container is observed for approximately 1 hour. The time that each insect spends on the rodent is recorded and number of lesions caused by the insect on the skin of the rodent is recorded. The insects spend less time on rodents receiving the test compositions than on the rodents receiving the control substance. The rodents receiving the test compositions receive fewer lesions than the rodents receiving the control substances.

Example 31 Repellent Effect of Compositions to Mosquitoes

About three cages are each stocked with about 100, southern house mosquitoes (culex quinquefasciatus), which are about 7 to 10 days-old. The mosquitoes are starved for about 12 hours. Each cage is supplied with four containers, each filled with cotton that has been soaked with sugar water.

Three of the four containers are treated randomly with about 1000 ppm (about 1 mg/l) of the composition being tested, while the remaining container serves as an untreated control. The containers are positioned in the four opposing corners of each cage and landing counts are conducted at about 0, 1, 2, 4, and 6 hour intervals following addition of the compositions being tested to the respective containers. The containers are removed from the cage between exposure intervals. Each exposure interval lasts for about 5 minutes.

The repellent effect of the compositions described in Table DD are tested using this method.

TABLE DD Ingredients Composition (% expressed by weight) EE 10% DEET, 45% LFO, 45% cumin oil AA 50% geraniol, 40% thyme oil, 10% lemon grass oil BB 50% LFO, 50% cumin oil

LFO, cumin oil, geraniol, thyme oil, and lemon grass oil are regarded as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA), and, as such, are exempt from EPA pesticide registration requirements.

The landing counts are conducted at about 0, 1, 2, 4, and 6 hour intervals following addition of the compositions, set forth in Table DD, to the respective containers. The landing counts are set forth in Table EE. Percent repellency is calculated using this data and is expressed in Table FF. At each exposure interval, the Compositions EE, AA and BB display almost 100% repellency. Even after 6 hours, the Compositions display 100% repellency against mosquitoes.

TABLE EE Landing Counts During Exposure Interval Exposure Time (hrs) 0 1 2 4 6 Total Control 36 26 30 13 6 111 Composition EE 0 1 1 0 0 2 Composition AA 0 0 0 1 0 1 Composition BB 0 0 0 0 0 0

TABLE FF % Repellency ((control − composition)/ control) × 100 Exposure Time (hrs) 0 1 2 4 6 Composition EE 100 96.2 96.7 100 100 Composition AA 100 100 100 92.3 100 Composition BB 100 100 100 100 100

Example 32 Methods of Testing Repellent Effect and Pesticidal Effect of Compositions Containing Plant Essential Oils on Red Ants

Pesticidal effect of various compositions containing plant essential oils on red ants is tested in the following manner. A paper disk is treated with about 20 μl of each of the composition being tested and the treated disks are each placed in a vial. An untreated paper disk is placed in a control vial. Also, a paper disk is treated with about 20 μl 100% DEET and placed in a vial to compare the pesticidal effect of the compositions to that of DEET, a known commercial insect control agent. About three red ants are introduced into each vial and the opening to the vials are closed with cotton to prevent the insects from escaping. The insect is exposed to the compositions for about one hour or less and mortality is recorded.

Repellent effect of various compositions containing plant essential oils on red ants is tested in the following manner. A paper disk is treated with about 200 μl of each composition and placed in a dish. An untreated paper disk is placed in a control dish. Also, a paper disk is treated with about 200 μl 100% DEET and placed in a dish to compare the repellant effect of the compositions to that of DEET. Red ants are introduced into each dish. Insect behavior and number of visits to the treated paper disk are monitored for about 5 minutes. The number of visits by a red ant to the paper disk is recorded.

Residuality, with regard to pesticidal effect and repellent effect, is tested by treating a paper disk with the composition being tested, keeping the treated paper disk under laboratory conditions for a predetermined period of time (e.g., 0 min, 6 hours, 1 day, 3 days, 5 days, 7 days), and exposing red ants to the treated paper disk in the above described manners.

Example 33 Repellent Effect and Pesticidal Effect of Compositions Containing Plant Essential Oils on Red Ants

The pesticidal effect and repellent effect of the compositions described in Table GG are tested using the methods described in Example 32. The untreated disks are neither toxic to nor do they repel red ants.

TABLE GG Ingredients Composition (% expressed by weight) Z 20% d-limonene, 10% lindenol, 10% eugenol, 10% phenylacetaldehyde, 50% geraniol AA 50% geraniol, 40% thyme oil, 10% lemon grass oil BB 50% LFO, 50% cumin oil CC 20% d-limonene, 20% thyme oil, 20% geraniol, 20% a- pinene, 20% p-cymene DD 10% DEET, 18% d-limonene, 18% thyme oil, 18% geraniol, 18% a-pinene, 18% p-cymene EE 10% DEET, 45% LFO, 45% cumin oil FF 44% LFO 44% cumin oil, 10% geraniol, 2% thyme oil

Each of the compositions results in 100% mortality, equivalent to that of DEET, when exposed to the paper disks about 0 min, 6 hours, 1 day, 3 days, 5 days, or 7 days after the paper disks are treated with the composition.

As shown in Table HH, red ants are repelled by the compositions used to treat the paper disks. Additionally, with regard to residuality, the compositions outperform DEET by retaining their potency for at least a week after being applied to the paper disks, while DEET begins to loose potency after 1 day. Table HH shows the number of trips by the red ants to the treated paper disks. The time periods set forth in the chart, 0 min, 6 hours, 1 day, 3 days, 5 days, or 7 days, refer to the approximate time elapsed between treatment of the paper disk with the composition and exposure of the red ants to the treated paper disk

TABLE HH 0 min 6 hours 1 day 3 days 5 days 7 days Composition Z 0 0 0 0 0 0 Composition AA 0 0 0 0 0 0 Composition BB 0 0 0 0 0 0 Composition CC 0 0 0 0 0 0 Composition DD 0 0 0 0 0 0 Composition EE 0 0 0 0 0 0 Composition FF 0 0 0 0 0 1 DEET (100%) 0 0 1 2 2 2

Example 34 Repellent Effect and Pesticidal Effect of Compositions Containing Plant Essential Oils on Red Ants

The pesticidal effect and repellent effect of the compositions described in Table JJ were tested using the methods described in Example 32. Treatment with each of the compositions caused a repellent effect and a pesticidal effect.

TABLE JJ Ingredients Composition (% expressed by weight) GG 10% d-limonene, 30% thyme oil, 35% geraniol, 10% a- pinene, 10% p-cymene, 5% phenylacetaldehyde HH 15% d-limonene, 50% geraniol, 15% a-pinene, 15% p- cymene, 5% phenylacetaldehyde JJ 50% d-limonene, 50% p-cymene KK 33.3% d-limonene, 33.3% p-cymene, 33.3% a-pinene LL 50% d-limonene, 50% thyme oil MM 50% thyme oil, 50% a-pinene NN 33.3% thyme oil, 33.3% a-pinene, 33.3% p-cymene OO 50% a-pinene, 50% p-cymene PP 25% linalool, 25% a-pinene, 25% p-cymene, 25% thyme oil QQ 33.3% linalool, 33.3% a-pinene, 33.3% p-cymene RR 33.3% d-limonene, 33.3% p-cymene, 33.3% thymol SS 25% d-limonene, 25% p-cymene, 25% thymol, 25% geraniol

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. It is intended that the Specification and Example be considered as exemplary only, and not intended to limit the scope and spirit of the invention. The references and publications cited herein are incorporated herein by this reference.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the Specification, Examples, and Claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the Specification, Example, and Claims are approximations that may vary depending upon the desired properties sought to be determined by the present invention. 

1. A method of pest control, comprising: providing a composition comprising at least two active ingredients; and contacting a pest with the composition, wherein the contacting results in a change in intracellular levels of cAMP, Ca²⁺, or both in said pest, and said change results in synergistic pest control.
 2. A method of pest control, comprising: providing a composition comprising at least two active ingredients, wherein at least one active ingredient is a ligand of a G-protein-coupled receptor in a target pest; and contacting the pest with the composition, wherein the contacting results in synergistic pest control.
 3. The method of claim 2, wherein only one active ingredient is a ligand of the G-protein-coupled receptor.
 4. The method of claim 2, wherein the at least one active ingredient is a ligand of a G-protein-coupled receptor selected from the group consisting of a pest tyramine receptor, and pest Or43a receptor, and a pest Or83b receptor.
 5. The method of claim 4, wherein only one active ingredient is a ligand of the G-protein-coupled receptor.
 6. The method of claim 2, wherein the at least one active ingredient is a ligand of at least two G-protein-coupled receptors selected from the group consisting of a pest tyramine receptor, and pest Or43a receptor, and a pest Or83b receptor.
 7. The method of claim 6, wherein only one active ingredient is a ligand of the at least two G-protein-coupled receptors.
 8. The method of claim 2, wherein the at least two active ingredients are selected from the group consisting of: black seed oil, camphene, carvacrol, d-carvone, l-carvone, 1,8-cineole, p-cymene, diethyl phthalate, geraniol, isopropyl citrate, lemon grass oil, lilac flower oil, lime oil, d-limonene, linalyl anthranilate, linalool, lindenol, methyl citrate, methyl di-hydrojasmonate, myrcene, perillyl alcohol, phenyl acetaldehyde, α-pinene, β-pinene, piperonal, piperonyl, piperonyl acetate, piperonyl alcohol, piperonyl amine, quinone, sabinene, α-terpinene, terpinene 900, gamma-terpineol, 2-tert-butyl-p-quinone, α-thujone, allyl sulfide, allyl trisulfide, allyl-disulfide, anethole, artemisia alcohol acetate, benzyl acetate, bergamotene, β-bisabolene, bisabolene oxide, α-bisabolol, bisabolol oxide, bisobolol oxide β, bornyl acetate, β-bourbonene, α-cadinol, α-campholene, α-campholene aldehyde, camphor, caryophyllene oxide, chamazulene, cinnamaldehyde, cis-verbenol, citral A, citral B, citronellol, citronellyl acetate, citronellyl formate, α-copaene, commint oil, β-costol, cryptone, curzerenone, davanone, diallyl tetrasulfide, dihydropyrocurzerenone, β-elemene, gamma-elemene, elmol, estragole, 2-ethyl-2-hexen-1-ol, eugenol acetate, α-farnesene, (Z,E)-α-farnesene, E-β-farnesene, fenchone, furanodiene, furanoeudesma-1,3-diene, furanoeudesma-1,4-diene, furano germacra 1,10(15)-diene-6-one, furanosesquiterpene, geraniol acetate, germacrene D, germacrene B, α-gurjunene, α-humulene, α-ionone, β-ionone, isoborneol, isofuranogermacrene, iso-menthone, iso-pulegone, jasmone, limonene, linalyl acetate, lindestrene, methyl-allyl-trisulfide, menthol, 2-methoxy furanodiene, menthone, menthyl acetate, methyl cinnamate, myrtenal neraldimethyl acetate, nerolidol, nonanone, 1-octanol, E ocimenone, Z ocimenone, 3-octanone, ocimene, octyl acetate, peppermint oil, α-phellandrene, β-phellandrene, prenal, pulegone, sabinyl acetate, α-santalene, santalol, sativen, δ-selinene, β-sesquphelandrene, spathulenol, tagetone, 4-terpineol, α-terpinolene, α-terpinyl acetate, α-thujene, thymyl methyl ether, trans-caryophyllene, trans-pinocarveol, trans-verbenol, verbenone, yomogi alcohol, zingiberene, and dihydrotagentone.
 9. The method of claim 1, wherein said synergistic pest control comprises a repellent effect.
 10. The method of claim 1, wherein said synergistic pest control comprises a pesticidal effect.
 11. The method of claim 1, wherein said composition further comprises at least one fixed oil.
 12. The method of claim 11, wherein the at least one fixed oil selected from the group consisting of castor oil, corn oil, cumin oil, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, and soy bean oil.
 13. The method of claim 1, wherein the at least two active ingredients comprise geraniol and thyme oil.
 14. The method of claim 13, wherein the at least two active ingredients additionally comprise d-limonene.
 15. The method of claim 14, wherein the at least two active ingredients additionally comprise piperonal.
 16. The method of claim 15, wherein the at least two active ingredients additionally comprise linalool.
 17. The method of claim 15, wherein the at least two active ingredients additionally comprise lime oil.
 18. The method of claim 1, wherein the at least two active ingredients comprise lilac flower oil and d-limonene.
 19. The method of claim 18, wherein the at least two active ingredients further comprise thyme oil.
 20. The method of claim 18, wherein the at least two active ingredients further comprise lime oil.
 21. The method of claim 1, wherein the at least two active ingredients comprise d-limonene, linalool, piperonal, and geraniol.
 22. The method of claim 1, wherein the at least two active ingredients comprise linalool, thymol, α-pinene, and p-cymene.
 23. The method of claim 23, wherein the at least two active ingredients further comprise t-anethole. 